Morphological studies

The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, P.R. China. The macromorphological descriptions are based on field notes. The colour terms follow Petersen [9]. Micromorphological data were obtained from the dried specimens, and observed under a light microscope (Nikon Eclipse E 100, Tokyo, Japan) following a previous study [10]. The following abbreviations were used for the microscopic characteristic descriptions: KOH = 5% potassium hydroxide, CB = cotton blue, CB– = acyanophilous, IKI = Melzer’s reagent, IKI– = both non-amyloid and non-dextrinoid, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens.

Molecular procedures and phylogenetic analyses

The CTAB rapid plant genome extraction kit DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from dried specimens, according to the manufacturer’s instructions, with some modifications: a small piece of dried fungal specimen (approximately 30 mg) was ground to a powder with liquid nitrogen. The powder was transferred to a 1.5 mL centrifuge tube, suspended in 0.4 mL of lysis buffer, and incubated in a 65°C water bath for 60 min. Then, 0.4 mL of phenol-chloroform (24:1) was added to the tube, and the suspension was shaken vigorously. After centrifugation at 13,000 rpm for 5 min, 0.3 mL of supernatant was transferred to a new tube and mixed with 0.45 mL of binding buffer. The mixture was then transferred to an adsorbing column (AC) for centrifugation at 13,000 rpm for 0.5 min. Then, 0.5 mL of inhibitor removal fluid was added to the AC, and the solution was centrifuged at 12,000 rpm for 0.5 min. After washing twice with 0.5 mL of washing buffer, the AC was transferred to a clean centrifuge tube, and 0.1 mL of elution buffer was added to the middle of the adsorbed film to elute the genomic DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 [11]. The PCR procedure for the ITS region was as follows: initial denaturation at 95°C for 3 min; followed by 35 cycles at 94°C for 40 s, 58°C for 45 s and 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company. All newly generated sequences were deposited in GenBank (Table 1).

Table 1

List of species, specimens, and GenBank accession number of sequences used in this study.

Species name	Sample no.	GenBank accession no.		References
		ITS	nLSU
Abortiporus biennis	EL 6503	JN649325	JN649325	[15]
Antrodia albida	CBS 308.82	DQ491414	DQ491414	[16]
A. heteromorpha	CBS 200.91	DQ491415	DQ491415	[16]
Antrodiella semisupina	X 242	JN710521	JN710521	[5]
Byssomerulius corium	FP 102382	KP135007	KP135230	[17]
Ceriporiopsis gilvescens	BRNM 710166	FJ496684	FJ496684	[18]
Climacocystis borealis	KH 13318	JQ031126	JQ031126	[6]
Coriolopsis caperata	LE(BIN) 0677	AB158316	AB158316	[18]
Daedalea quercina	Miettinen 12662	JX109855	JX109855	[6]
Earliella scabrosa	PR 1209	JN165009	JN165009	[19]
Fomitopsis pinicola	CCBAS 536	FJ608588	—	[20]
F. rosea	ATCC 76767	DQ491410	DQ491410	[16]
Fragiliporia fragilis	Dai 13080	KJ734260	KJ734260	[21]
F. fragilis	Dai 13559	KJ734261	KJ734261	[21]
F. fragilis	Dai 13561	KJ734262	KJ734262	[21]
Ganoderma lingzhi	Wu 100638	JQ781858	—	[22]
Gelatoporia subvermispora	BRNU 592909	FJ496694	FJ496694	[18]
Grammothelopsis subtropica	Cui 9035	JQ845094	JQ845097	[22]
Heterobasidion annosum	PFC 5252	KC492906	KC492906	[6]
Hornodermoporus martius	MUCL 41677	FJ411092	FJ411092	[23]
Hypochnicium lyndoniae	NL 041031	JX124704	JX124704	[6]
Irpex lacteus	DO 421/951208	JX109852	JX109852	[6]
Junghuhnia crustacea	X 262	JN710553	JN710553	[5]
Mycoacia fuscoatra	KHL 13275	JN649352	JN649352	[15]
M. nothofagi	KHL 13750	GU480000	GU480000	[24]
Obba rivulosa	KCTC 6892	FJ496693	FJ496693	[18]
O. valdiviana	FF 503	HQ659235	HQ659235	[25]
Perenniporia medulla-panis	MUCL 43250	FJ411087	FJ411087	[23]
P. ochroleuca	MUCL 39726	FJ411098	FJ411098	[23]
P. chrysocreas	KUC 2012112324	KJ668482	KJ668482	[17]
Phlebia fuscotuberculata	CLZhao 10239	MT020760	MT020738	[26]
P. hydnoidea	HHB 1993	KY948778	KY948778	[7]
P. radiata	AFTOL 484	AY854087	AY854087	[27]
P. tomentopileata	CLZhao 9509	MT020762	MT020740	[26]
P. tongxiniana	CLZhao 5217	MT020778	MT020756	[26]
P. tremellosa	ES 20082	JX109859	JX109859	[6]
Piloporia sajanensis	Mannine 2733a	HQ659239	HQ659239	[18]
Podoscypha venustula	LR 40821	JX109851	JX109851	[6]
Polyporus tuberaster	CulTENN 10197	AF516596	AF516596	[6]
Sebipora aquosa	Miettinen 8680	HQ659240	HQ659240	[25]
Skeletocutis amorpha	Miettinen 11038	FN907913	FN907913	[18]
S. jelicii	H 6002113	FJ496690	FJ496690	[18]
S. portcrosensis	LY 3493	FJ496689	FJ496689	[18]
S. subsphaerospora	Rivoire 1048	FJ496688	FJ496688	[18]
Steccherinum autumnale	VS 2957	JN710549	JN710549	[5]
S. bourdotii	RS 10195	JN710584	JN710584	[5]
S. collabens	KHL 11848	JN710552	JN710552	[5]
S. fimbriatellum	OM 2091	JN710555	JN710555	[5]
S. fimbriatum	KHL 11905	JN710530	JN710530	[5]
S. formosanum	TFRI 652	EU232184	EU232268	[8]
S. lacerum	TN 8246	JN710557	JN710557	[5]
S. meridionalis	MR 10466	KY174994	KY174994	[8]
S. meridionalis	MR 11086	KY174993	KY174993	[8]
S. meridionalis	MR 284	KY174992	KY174992	[8]
S. neonitidum	MCW 371/12	KY174990	KY174990	[8]
S. neonitidum	RP 79	KY174991	KY174991	[8]
S. nitidum	KHL 11903	JN710560	JN710560	[5]
S. nitidum	MT 33/12	KY174989	KY174989	[8]
S. nitidum	FP 105195	KP135323	KP135227	[17]
S. ochraceum	KHL 11902	JN710590	JN710590	[5]
S. polycystidiferum	RP 140	KY174996	KY174996	[8]
S. polycystidiferum	MCW 419/12	KY174995	KY174995	[8]
S. pseudozilingianum	MK 1004	JN710561	JN710561	[5]
S. robustius	G 1195	JN710591	JN710591	[5]
S. tenue	KHL 12316	JN710598	JN710598	[5]
S. tenuispinum	OM 8065	JN710599	JN710599	[5]
S. tenuispinum	LE 231603	KM411452	KM411452	[8]
S. tenuispinum	VS 2116	JN710600	JN710600	[5]
S. tenuissimum	CLZhao 894	MW204581	MW204570	this study
S. tenuissimum	CLZhao 3153	MW204582	MW204571	this study
S. tenuissimum	CLZhao 4294	MW204583	MW204572	this study
S. tenuissimum	CLZhao 5100	MW204584	MW204573	this study
S. undigerum	MCW 426/13	KY174986	KY174986	[8]
S. undigerum	MCW 472/13	KY174987	KY174987	[8]
S. undigerum	MCW 496/14	KY174988	KY174988	[8]
S. xanthum	CLZhao 4381	MW204585	MW204574	this study
S. xanthum	CLZhao 4479	MW204586	MW204575	this study
S. xanthum	CLZhao 5024	MW204587	MW204576	this study
S. xanthum	CLZhao 5030	MW204588	MW204577	this study
S. xanthum	CLZhao 5032	MW204589	MW204578	this study
S. xanthum	CLZhao 5044	MW204590	MW204579	this study
S. xanthum	CLZhao 8124	MW204591	MW204580	this study
Stereum hirsutum	NBRC 6520	AB733150	AB733325	[18]
Tyromyces chioneus	Cui 10225	KF698745	KF698745	[28]

Sequencher 4.6 (GeneCodes, Ann Arbor, MI, USA) was used to edit the DNA sequences. The sequences were aligned in MAFFT 7 (http://mafft.cbrc.jp/alignment/server/) using the “G-INS-I” strategy and manually adjusted in BioEdit [12]. The sequence alignment was deposited in TreeBase (submission ID 27218). Sequences of Heterobasidion annosum (Fr.) Bref. and Stereum hirsutum (Willd.) Pers. obtained from GenBank was used as an outgroup to root trees following previous study [6] in the ITS + nLSU analysis (Fig 1), and Byssomerulius corium (Pers.) Parmasto and Irpex lacteus (Fr.) Fr. were used as an outgroup in the ITS + nLSU (Fig 2) analyses following previous study [8].

Fig 1

Maximum parsimony strict consensus tree illustrating the phylogeny of two new species and related species in Polyporales based on ITS + nLSU sequences.

Branches are labelled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.95 respectively. Clade names follow Binder et al. [6].

Fig 2

Maximum parsimony strict consensus tree illustrating the phylogeny of two new species and related species of Steccherinum based on ITS + nLSU sequences.

Branches are labelled with maximum likelihood bootstrap values higher than 70%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.95.

Maximum parsimony analyses were applied to the ITS + nLSU dataset sequences. The approaches used for the phylogenetic analysis followed previous study [13], and the tree construction procedure was performed in PAUP* version 4.0b10 [14]. All characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees was set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [29]. Descriptive tree statistics, including tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimony tree generated. The sequences were also analysed using maximum likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org) [30]. Branch support (BS) for the ML analysis was determined by 1000 bootstrap replicates.

MrModeltest 2.3 [31] was used to determine the best-fit evolution model for each dataset through Bayesian inference (BI). Bayesian inference was calculated with MrBayes 3.1.2, with a general time reversible (GTR+I+G) model of DNA substitution and gamma distribution rate variation across sites [32]. Four Markov chains were run for 2 runs from random starting trees for 800 thousand generations (Fig 1), for 1100 thousand generations (Fig 2) and trees were sampled every 100 generations. The first one-fourth of the generations were discarded as burn-in. A majority rule consensus tree of all the remaining trees was calculated. The branches were considered significantly supported if they received maximum likelihood bootstrap values (BS) >75%, maximum parsimony bootstrap values (BT) >75%, or Bayesian posterior probabilities (BPP) >0.95.

Nomenclature acts

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Molecular phylogeny

The ITS + nLSU dataset (Fig 1) included sequences from 49 fungal specimens representing 45 species. The dataset had an aligned length of 1820 characters, of which 995 characters are constant, 202 are variable and parsimony-uninformative, and 623 are parsimony-informative. The maximum parsimony analysis yielded 2 equally parsimonious trees (TL = 4151, CI = 0.3404, HI = 0.6596, RI = 0.5438 and RC = 0.1851). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, rates = invgamma, prset statefreqpr = dirichlet (1,1,1,1)). The Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies of 0.006728 (BI).

The phylogenetic tree (Fig 1) inferred from the ITS + nLSU sequences, demonstrated seven major clades for 45 sampled species in Polyporales. Two new Steccherinum species nested into the residual polyporoid clade. Steccherinum xanthum grouped with S. tenuissimum and was closely related to S. robustius (J. Erikss. & S. Lundell) J. Erikss.

The ITS + nLSU dataset (Fig 2) included sequences from 40 fungal specimens representing 21 species. The dataset had an aligned length of 2117 characters, of which 1587 characters are constant, 151 are variable and parsimony-uninformative, and 379 are parsimony-informative. The maximum parsimony analysis yielded 576 equally parsimonious trees (TL = 1322, CI = 0.551, HI = 0.449, RI = 0.798 and RC = 0.440). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, rates = invgamma, prset statefreqpr = dirichlet (1,1,1,1)). The Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies of 0.009054 (BI).

The phylogenetic tree (Fig 2) inferred from the ITS + nLSU sequences had 19 species of Steccherinum and revealed that Steccherinum tenuissimum and S. xanthum were sister to S. robustius with high support (100% BP, 100% BS and 1.00 BPP). Steccherinum tenuissimum and S. xanthum formed a well-supported monophyletic lineage distinct from other Steccherinum species.

Taxonomy

Steccherinum tenuissimum C.L. Zhao & Y.X. Wu, sp. nov. Figs 3 and 4

Fig 3

Basidiomata of Steccherinum tenuissimum.

Bars: A = 1 cm, B = 1 mm (holotype).

Fig 4

Microscopic structures of Steccherinum tenuissimum (drawn from the holotype).

A: Basidiospores. B: Basidia and basidioles. C: Cystidia. D: A section of hymenium. Bars: A = 5 μm, B–D = 10 μm.

MycoBank No.: MB 837795

Holotype: China. Yunnan Province, Pu’er, Laiyanghe National Forest Park, on fallen angiosperm branch, 30 September 2017, CLZhao 3153 (SWFC).

Etymology: tenuissimum (Lat.): referring to the relatively thin basidiomata.

Basidiomata: Annual, adnate, without odour or taste when fresh, becoming membranaceous up on drying, very thin, up to 20 cm long, 3 cm wide, 50–100 μm thick. Hymenial surface odontioid, with round aculei, 3–4 per mm, 0.2–0.5 mm long, white to cream when fresh, turning to cream to olivaceous buff upon drying.

Hyphal structure: Hyphal system dimitic, generative hyphae with clamp connections, colourless, thin-walled, branched, interwoven, 1.8–3.5 μm in diameter, IKI–, CB–; skeletal hyphae colourless, thick-walled, unbranched, 2.4–4.5 μm in diameter, IKI–, CB+; tissues unchanged in KOH.

Hymenium: Cystidia numerous, colourless, strongly encrusted, 22–39 × 4.5–6 μm; cystidioles absent. Basidia subclavate, with 4-sterigmata and basal clamp connections, 9.5–19 × 2.5–5.5 μm; basidioles dominant, in shape similar to basidia, but slightly smaller.

Spores: Basidiospores ellipsoid, colourless, thin-walled, smooth, with oil drops, IKI–, CB–, 3–5(–5.5) × 2–3.5 μm, L = 4.16 μm, W = 2.86 μm, Q = 1.40–1.52 (n = 120/4).

Additional specimens (paratypes) examined: China. Yunnan Province, Yuxi, Xinping County, Mopanshan National Forest Park, on fallen angiosperm branch, 16 January 2017, CLZhao 894 (SWFC); Xinping County, the Ancient Tea–Horse Road, on fallen angiosperm branch, 13 January 2018, CLZhao 5100 (SWFC); Pu’er, Jingdong County, Wuliangshan National Nature Reserve, on fallen branch of Pinus, 5 October 2017, CLZhao 4294 (SWFC).

Steccherinum xanthum C.L. Zhao & Y.X. Wu, sp. nov. Figs 5 and 6

Fig 5

Basidiomata of Steccherinum xanthum.

Bars: A = 1 cm, B = 1 mm (holotype).

Fig 6

Microscopic structures of Steccherinum xanthum (drawn from the holotype).

A: Basidiospores. B: Basidia and basidioles. C: Cystidia. D: A section of hymenium. Bars: A = 5 μm, B–D = 10 μm.

MycoBank No.: MB 837796

Holotype: China. Yunnan Province, Pu’er, Jingdong County, Wuliangshan National Nature Reserve, on fallen angiosperm branch, 6 October 2017, CLZhao 4479 (SWFC).

Etymology: xanthum (Lat.): referring to the buff hymenial surface of the type specimen.

Basidiomata: Annual, resupinate, adnate, without odour or taste when fresh, becoming membranaceous up on drying, up to 10 cm long, 4 cm wide, 100–200 μm thick. Hymenial surface odontioid, with round aculei, 5–6 per mm, 0.1–0.3 mm long, white to cream when fresh, turning buff to yellow upon drying.

Hyphal structure: Hyphal system monomitic, generative hyphae with clamp connections, colourless, thin-walled, branched, covered by crystals, interwoven, 2–4.5 μm in diameter; IKI–, CB–; tissues unchanged in KOH.

Hymenium: Cystidia numerous, strongly encrusted in the apical part, 35.5–125 × 5–9 μm; cystidioles absent. Basidia clavate, with 4-sterigmata and basal clamp connections, 10–19.3 × 3–5.2 μm, basidioles dominant, in a shape similar to basidia, but slightly smaller.

Spores: Basidiospores ellipsoid, colourless, smooth, thin-walled, with oil drops, IKI–, CB–, 2.7–5(–5.6) × 2–3.9(–4.4) μm, L = 3.48 μm, W = 2.63 μm, Q = 1.25–1.41 (n = 240/9).

Additional specimens (paratypes) examined: China. Yunnan Province, Pu’er, Zhenyuan County, Heping town, Jinshan Forest Park, on fallen angiosperm trunk, 12 January 2018, CLZhao 5024, 5032, 5044 (SWFC); 21 August 2018, CLZhao 8124 (SWFC); on fallen angiosperm branch 12 January 2018, CLZhao 5030 (SWFC); Jingdong County, Wuliangshan National Nature Reserve, on fallen angiosperm trunk, 6 October 2017, CLZhao 4381 (SWFC).